Adeno-associated virus (AAV) is a leading candidate vector for in vivo and ex vivo gene therapy, for cellular delivery of DNA to correct genetic errors deficiencies or predispositions to disease. Our long term goal is to understand the virus-host interactions that mediate the efficiency with which AAV enters specific cells and tissues. This foundation will support the widely sought goals of targeting naturally non-permissive cells, and reducing the adverse effects of transgene expression in off-target tissues. Aim 1 focuses on the first step in cell entry, attachment to the primary receptor, which is heparan sulfate (HS) proteoglycan for several human AAVs. It builds on our structures of AAV-2, -3 & -6, and unexpected diversity in the HS binding site of these otherwise homologous viruses. Crystallography and electron microscopy (EM) will be used to characterize the diversity of HS interactions that has evolved in AAV, correlating structure with measured heparin affinity, cell attachment and cell entry, measured by in vitro transgene expression. Our goal is to elucidate the structure-affinity determinants of attachment that modulate the efficiency of entry and the differential susceptibility of specific cell types. Our understanding will be tested by engineering vectors with altered HS interactions, and characterizing their attachment and entry in a panel of representative cell lines. Aim 2 will elucidate the major antigenic determinants in a human polyclonal neutralizing antibody response. Our structure of AAV-DJ, a chimeric recombinant selected to escape pooled neutralizing serum, shows change localized around a likely dominant AAV2 monoclonal epitope visualized by EM. It opens a direct path to human polyclonal interactions: selection for neutralizing serum escape from a library of random single-site mutants. Dominant antigenic sites will be mapped through clustering of mutations on the AAV structure. Aim 2 will have impact in the design of neutralization-resistant vectors for in vivo human gene therapies, and will guide structural studies of MAb complexes to those that best model interactions that are important in human patients. Aim 3 focuses on co-receptors involved in the second step of cell entry. Our first goal is to rationalize a growing list of integral membrane proteins that have been implicated by various lines of evidence. Genomic approaches, including gene trapping, cDNA expression cloning and RNA interference, will be used to rank the functional importance of interactions to viral entry. They will also help distinguish direct AAV interactions from modulation of cellular pathways indirectly affecting rAAV transgene expression. Following confirmation by complementation and other methods, ecto-domain constructs will be expressed for measurement of binding affinities and for structural characterization by cryo-EM or footprinting by hydrogen / deuterium exchange mass spectrometry. Thus, Aim 3 will advance the basic understanding of a poorly understood aspect of AAV biology that will likely also prove important in cellular targeting of vectors.